The lack of effective treatments for many cancers and the occurence of resistance to available therapies make it critically important to identify new targets and strategies for intervention against a number of human malignancies. This multi-PI-directed study will set out to establish the importance of previously unrecognized, reversible protein posttranslational modifications (lysine succinylation and malonylation) in the development of the malignant state. The studies described in this application arise from two exciting new research developments. The first is based on our very recent discovery suggesting the existence of novel post-translational protein modifications on several metabolic enzymes in cells. These modifications appear to be regulated by the mitochondrial protein Sirt5, a member of the Sir2 family of proteins (for Silent information regulator 2). The second involves the renewed attention being directed toward understanding the roles played by cellular metabolism in cancer progression. This is especially the case for the enhanced glycolytic activity (the Warburg effect) and markedly elevated glutamine metabolism (i.e. glutamine addiction) exhibited by cancer cells. When combined with our preliminary findings suggesting that Sirt5 is required for the transformed phenotypes of a number of human cancer cells, these different pieces of evidence have led us to put forward the novel hypothesis that the desuccinylation and/or demalonylation of key enzymes in cancer metabolism are essential for satisfying the biosynthetic and bioenergetic requirements of malignant transformation. We will test this hypothesis in cell culture experiments (Aim 1) and mouse models (Aim 2), as well as delineate the molecular mechanisms underlying the requirement of demalonylation/desuccinylation for the malignant state of cancer cells (Aim 1). Moreover, we believe that targeting Sirt5 and blocking its ability to regulate these post-translational modifications in cancer cells will offer new approaches for intervention against this disease. We will identify novel small molecule inhibitors that specifically target Sirt5 (Aim 3) and demonstrate their effectiveness at blocking malignant transformation in cell culture (Aim 1) and in a mouse model of Kras-induced lung cancer (Aim 2), a malignancy for which the development of new therapeutic strategies is especially needed. While a subset of lung cancers can be treated successfully with EGF receptor inhibitors, the approximately 25-50% of lung adenocarcinomas harboring Kras mutations are resistant to these inhibitors as well as chemotherapy, and clinically effective drugs targeting Kras have remained elusive. Thus, we feel that developing strategies that target these novel Sirt5-regulated post- translational modifications as a means to help 're-set' the altered metabolism of cancer cells represents a transformative approach that could ultimately prove beneficial to lung cancer patients, as well as provide a general therapeutic benefit against a broad range of human malignancies, given the fundamental nature of metabolic alterations in cancer.